(a) Field of the Invention
The present invention relates to therapies, diagnostics and research reagents for disease states, which respond to the alteration of cyclin E gene expression, a gene involved in the cell cycle. More particularly, the invention relates to the use of antisense oligonucleotides, which hybridizes to a nucleic acid sequence coding for the cyclin E gene. The invention also relates to a method for preventing restenosis or for treating pathologies, which involves cyclin E gene, such as vascular proliferative diseases or other proliferative disorders such as psoriasis, cancer and related metastasis.
(b) Description of Prior Art
Cell duplication of mammalian cells is regulated by a large number of genes by which their expression of functions responds to mitogenic stimuli (Lanahan A. et al., Mol. Cell. Biol., 12:3919-3929, 1992). Cyclins are prime regulators of cell proliferation, which control the progression of cells through the cell cycle. They function by forming a complex with a class of protein kinases, i.e. cyclin-dependent kinases that are essential for cell cycle transitions (Nigg E A., Bioessays, 17(6):471-80, 1995). Normal quiescent cells are in the initial G0 phase. Cells enter the cell cycle under mitogenic stimulation via the G1 phase whereas cyclin D plays a regulatory role to ensure progression through the phase. Cyclin E regulates the entry of cells into the S phase and cyclin A ensures the progression through the S phase. Cyclins A and B ensure the progression through the G2 and M phases of the cell cycle, respectively.
Dysregulation of the G1 phase cyclins, more specifically cyclin E, has been implicated in abnormal cell proliferation. For example, cyclin E overexpression has been reported in rat esophageal tumorigenesis (Wang Q-S et al., Carcinogenesis, 17(8): 1583-1588, 1996), in the formation of human hepatic tumors (Tsuji T. et al. Biophys. Res. Comm., 242: 317-321, 1998), in ovarian cancer (Marone M. et al., Int. J. Cancer, 75:34-39), in breast cancer (Keyomarsi K et al., Oncogene, 11:941-950, 1995), colorectal carcinoma (Leach F. S. et al., Cancer Res., 53:1986-1989, 1993), gastric carcinoma (Akama Y. et al., Jpn. J. Cancer Res., 86:617-621, 1995) and acute lymphoblastic leukemia (Scuderi R. et al., Blood, 87:3360-3367, 1996).
Cyclin E is also implicated in abnormal cell proliferation following percutaneous transluminal angioplasty (PCTA) (Wei G L. et al., Circ. Res., 80:418-426, 1997). PCTA is an accepted form of treatment of coronary and peripheral vascular disease. Since its introduction in 1977 for the treatment for coronary disease, primary success rates have reached very high levels (90% to 95%) and complication rates of 1% to 5% are now the standards. However, it was observed that narrowing of the dilated vessel would reoccur at the same site within three to six months following the procedure. The incidence of restenosis following balloon angioplasty may be as high as 55% and 65% in the coronary and peripheral arteries, respectively. All pharmacological approaches to prevent the occurrence of restenosis have failed.
A number of mechanical alternatives to balloon angioplasty have been developed and investigated. However, none of these alternatives have yet shown to diminish conclusively the incidence of restenosis following percutaneous revascularization, except for a modest reduction obtained with the Palmaz-Schatz stent in selected patients. This effect is explained by the propensity of the stent to achieve a consistently greater increase in lumen diameter immediately after the procedure by limiting the phenomenon of elastic recoil. Although many of the risk factors for restenosis have been identified, most of them are difficult to influence.
PCTA results in unavoidable vessel wall injury. Disruption of endothelial and vessel wall structure triggers molecular and cellular events, which leads in some patients to restenosis. Several growth factors, cytokines and cell-surface receptors have been implicated in the proliferation process. In animal models of vascular injury, following the immediate loss of lumen diameter accounted by elastic recoil, an important cascade of events leads to smooth muscle cell (SMC) proliferation that begins 24 hours post-angioplasty. SMC proliferation appears to be a consistent response to balloon dilatation and/or denudation of the artery. Cell replication has been reported to peak within seven days after the angioplasty. Twenty-eight days after the angioplasty, SMC proliferation in the media as well as in the intima appears normalized. This process is then followed by matrix deposition over the next several weeks.
A line of therapy of treatment of uncontrolled cellular proliferation involves radiotherapy. For example, cancerous tumors are treated with radiation therapy, either by external radiation or by applying the radioactive source internally. Another example is the use of a radioactive wire, catheter, stent or balloon that may be applied to an artery undergoing a PCTA procedure. Recently, a procedure involving the infiltration of a radiolabeled oligonucleotide into the vessel wall has been proposed (U.S. Pat. No. 5,821,354).
Pharmacological compounds have been extensively used for cancer therapy with success in a wide array of cancer subtypes. However, these compounds have not proven to succeed in reducing restenosis.
A new avenue of treatment of arteries undergoing PCTA is to locally deliver drugs. In a rat model, antisense oligonucleotides directed against proliferating-cell nuclear antigen (PCNA) (Simons M. et al., J. Clin. Invest., 93(6):2351-2356, 1994) inhibits the SMC proliferation into the intima. The oligonucleotide was mixed in a Pluronic gel that was applied to the artery following the PCTA procedure. Other studies involved the use of antisense c-myb, c-myc and CDK2 kinase oligonucleotides.
Villa and colleagues (Villa A E et al., Circ. Res., 76(4): 505-513, 1995) have unsuccessfully tried to use antisense c-myb oligonucleotides to inhibit restenosis following PCTA in the rat model. They reported that the presence of four contiguous guanine residues might be associated with an aptamer effect, which can be differentiated from a hybridization-dependent antisense mechanism.
Studies have also investigated c-myc antisense oligonucleotides in the prevention of restenosis. Shi and colleagues (Shi Y. et al., Circulation, 90 (2): 944-951, 1994) reported that they have successfully reduced smooth muscle proliferation and extracellular matrix accumulation in the lumen of the porcine coronary artery. However, clinical trials using this therapy was deemed unsuccessful (Holt C. M., Antisense Oligonucleotides for the treatment of coronary restenosis. Antisense 98, London. Oct. 8-9, 1998). Whether the oligonucleotide or the transport delivery device was in fault was not determined.
Other studies have examined the effects of antisense CDK2 oligonucleotides in the prevention of neointimal formation in murine coronary allografts (Suzuki J.-I. et al., Nat. Med., 3(8):900-903, 1997). They reported that intraluminal administration of antisense CDK2 kinase oligonucleotides, a cell cycle regulatory gene, could inhibit neointimal formation after cardiac transplantation.
However, there are no studies up to date that have evaluated the use of antisense oligonucleotides targeting a cell cycle in the reduction of neointimal proliferation. A previous study has shown that certain genes implicated in the cell cycle progression were induced following a PCTA (Wei G L. et al., Circ. Res., 80:418-426, 1997). Indeed, rat arteries subjected to PCTA do express in the following days CDK2, PCNA and cyclin E and A gene products.
The role of cyclin E is to push the cells from the G1 phase of the cell cycle to the S phase, where cells are committed to divide. To date, there is no studies involving antisense constructs nor oligonucleotides that have been reported to inhibit the cyclin E gene product.
It would be highly desirable to be provided with a more effective method and a pharmaceutical composition for preventing uncontrolled cell proliferation, such as restenosis, cancer and psoriasis.
One aim of the present invention is to provide a more effective method for preventing uncontrolled cell proliferation, such as restenosis.
Another aim of the present invention is to provide a pharmaceutical composition for preventing uncontrolled cell proliferation.
In accordance with the present invention there is provided an antisense oligonucleotide for inhibiting cellular proliferation, said oligonucleotide being complementary to a 5xe2x80x2 untranslated region (5xe2x80x2-UTR) or to a 3xe2x80x2 untranslated region (3xe2x80x2-UTR) of cyclin E gene for inhibiting the expression of said cyclin E gene, thus inhibiting cellular proliferation.
Preferably, the antisense oligonucleotide has a nucleic acid sequence derived from SEQ ID NO:1 or SEQ ID NO:2.
The cellular proliferation may either be restenosis, or may be caused by a cancer or by psoriasis.
Also in accordance with the present invention there is provided a pharmaceutical composition comprising an antisense oligonucleotide as defined above, in combination with a pharmaceutically acceptable carrier.
Further in accordance with the present invention, there is provided a method for preventing cellular proliferation comprising the step of administering to a patient an antisense oligonucleotide complementary to a 5xe2x80x2 untranslated region (5xe2x80x2-UTR) or to a 3xe2x80x2 untranslated region (3xe2x80x2-UTR) of cyclin E gene for inhibiting the expression of said cyclin E gene, thus inhibiting cellular proliferation. The antisense oligonucleotide, as described above, may have a nucleic acid sequence derived from SEQ ID NO:1 or SEQ ID NO:2.
The antisense oligonucleotide is preferably delivered at a site of dilatation of an artery, in case of restenosis.
Also in accordance with the present invention, there is provided an antisense oligonucleotide for inhibiting cellular proliferation. The antisense oligonucleotide, as described above, is complementary to a 5xe2x80x2 untranslated region (5xe2x80x2-UTR) or to a 3xe2x80x2 untranslated region (3xe2x80x2-UTR) of cyclin E gene for inhibiting the expression of said cyclin E gene, thus inhibiting cellular proliferation.